Immunomagnetic separation of subpopulations of apolipoprotein A-I.

OBJECTIVE: This study was undertaken to measure the subfractions of high-density lipoprotein (HDL) in patients with diabetes or coronary artery disease and in normal control subjects. DESIGN: A new immunomagnetic separation technique was used to characterize the lipid profile in four groups: (1) control subjects, (2) patients with diabetes but no coronary artery disease (CAD), (3) those with CAD only, and (4) those with both diabetes and CAD. MATERIAL AND METHODS: To study the individual roles of the two discrete HDL subpopulations of particles--LpAI/AII (apolipoprotein [apo] A-I associated with A-II) and LpAI (apo A-I without A-II)--in lipoprotein metabolism, we developed an immunomagnetic separation technique using magnetic beads coated with antibodies to human apo A-II. The beads bind particles that contain both apo A-II and apo A-I and are precipitated by a magnetic field. LpAI levels were measured in the supernatant by performing an apo A-I radioimmunoassay. LpAI/AII levels were determined by subtracting the LpAI levels from total plasma apo A-I. RESULTS: In comparison with control subjects, patients with diabetes, CAD, or both had significantly decreased levels of LpAI/AII. LpAI levels were normal in patients with diabetes without CAD but significantly lower than control values in those with diabetes and CAD. CONCLUSION: Our findings suggest that both subpopulations of HDL particles have implications in the development of atherosclerosis in patients with and without diabetes.

A particle concentration fluorescence immunoassay for Lp(a).

The quantitation of Lp(a) by immunoassay presents a major technical problem, because the molecular mass of the (a) protein of Lp(a) can vary between 419,000 and 838,000 Da (Gaubatz et al. (1990) J. Lipid Res. 31, 603-612), and this variability is determined by at least 24 alleles of the (a) gene. In an attempt to overcome this problem, we have developed an assay that is independent of variation of the size of (a). The assay utilizes a mixture of monoclonal antibodies to (a) which do not react to plasminogen or to apolipoprotein (apo) B. These antibodies are bound to inert microscopic beads to capture the Lp(a) particles. Subsequently, a fluorescein-labeled monoclonal antibody to apo B is used for detection and quantitation. The assay is done with special microtiter plates containing filters so that the particles can be thoroughly washed after capture on the microbeads. Because Lp(a) particles contain only one apo B particle and the molecular weight of apo B is constant, the assay is not affected by variation in the size of apo(a). By binding the mixture of monoclonal antibodies to inert beads, it is possible to greatly increase the amount of antibody bound to an exposed surface and thus increase the sensitivity of the assay. A mixture of monoclonal antibodies can be used to increase the affinity of the capture step of the assay. The assay can be completed in 4 h and has a wide working range.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of human plasma apolipoproteins C-I, C-II, and C-III by radioimmunoassays.

We have developed radioimmunoassays for the quantification of apolipoproteins (apo) C-I, C-II, and C-III in human plasma. The apo C proteins were isolated from very low-density lipoproteins of patients with hypertriglyceridemia, fractionated on a Sephacryl column, and purified by diethylaminoethyl cellulose anion-exchange chromatography followed by reverse-phase fast protein liquid chromatography. The assays were sensitive, specific, and reproducible, and the standards demonstrated parallel immunoreactivity with plasma samples. Patients with hypertriglyceridemia (triglyceride level more than 2,200 mg/liter)--14 patients with diabetes and 12 with type V hyperlipoproteinemia--were compared with age- and sex-matched control subjects. In comparison with the control groups, levels of apoproteins C-I, C-II, and C-III were significantly increased in both disease groups, but the ratios of the C peptides to triglycerides were significantly lower, an indication of a relative deficiency of C apoproteins in hypertriglyceridemic states. Independent radioimmunoassays for each of the C apolipoproteins would help to study their individual roles in triglyceride-rich lipoprotein metabolism.

A rapid apolipoprotein E radioimmunoassay using solid-phase staphylococcus protein. Use of pooled plasma as a secondary standard.

A rapid apolipoprotein E (apo E) radioimmunoassay, which requires a total of 24 hour incubation as compared to the usual 3-5 days, has been developed in our laboratory. Solid phase staphylococcus protein A was used to separate bound and unbound labeled antigen. Use of a pooled plasma (quality control sample) as a secondary standard to reduce interassay variation was also described.